title: Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects.
abstract: Argonaute (Ago) proteins form the core of RNA-induced silencing complexes (RISCs) and mediate small RNA-guided gene silencing. In RNAi, short interfering RNAs (siRNAs) guide RISCs to complementary target RNAs, leading to cleavage by the endonuclease Ago2. Noncatalytic Ago proteins, however, contribute to RNAi as well but cannot cleave target RNA and often generate off-target effects. Here we show that synthetic siRNA duplexes interact with all Ago proteins, but a functional RISC rapidly assembles only around Ago2. By stabilizing the siRNA duplex, we show that the noncatalytic Ago proteins Ago1, -3, and -4 can be selectively blocked and do not form functional RISCs. In addition, stabilized siRNAs form an Ago2-RISC more efficiently, leading to increased silencing activity. Our data suggest novel parameters for the design of siRNAs with selective activation of the endonuclease Ago2.
authors.affiliation: University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Petri
authors.forename: Anne
authors.initials: A
authors.lastname: Dueck
authors.forename: Gerhard
authors.initials: G
authors.lastname: Lehmann
authors.forename: Nicholas
authors.initials: N
authors.lastname: Putz
authors.forename: Sabine
authors.initials: S
authors.lastname: Rüdel
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kremmer
authors.forename: Gunter
authors.initials: G
authors.lastname: Meister
mesh_terms: D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000378:metabolism*
chemical_list: C115848:AGO1 protein, human; C466422:AGO2 protein, human; C517143:AGO3 protein, human; C517144:AGO4 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex